Adenosylmethionine synthetase from streptomyces sp., gene sequences coding the same and method for mass production of secondary metabolites including antibiotics thereof

ABSTRACT

Disclosed is an isolated nucleotide sequence encoding an enzyme catalyzing biosynthesis of SAM (SAM-s) and its amino acid sequence. Also, the present invention provides a method for mass production of a useful secondary metabolite including antibiotics using the isolated nucleotide sequence and SAM, where SAM acts as a methyl group donor.

TECHNICAL FIELD

The present invention relates to S-adenosyl-L-methionine synthetase and a nucleotide sequence encoding the same, and more particularly, to an isolated nucleotide sequence encoding an enzyme catalyzing biosynthesis of SAM (SAM-s) from adenosyl triphosphate (ATP) and methionine, and its amino acid sequence. Also, the present invention is concerned with a method for mass production of a useful secondary metabolite including antibiotics using the nucleotide sequence and SAM.

PRIOR ART

S-adenosyl-L-methionine (SAM) is well known to play a critical role in cell growth and differentiation, essential for survival of living organisms including human beings. In living cells, SAM acts as a methyl group donor as well as a precursor for an amninopropyl group in a biosynthesis pathway of polyamine, where the methyl group and the polyamine are utilized in primary and secondary metabolisms.

It has been reported that SAM positively or negatively affects growth of bacteria including E. coli and Bacillus subtilis, thus causing their life cycles to change in a manner of inhibiting cell growth or stimulating morphological differentiation.

In addition, the biological function of SAM is also found to be essential for primary and secondary metabolisms in plants and animals. Especially, it has been reported that SAM as a methyl group donor affects differentiation, causing morphological changes in plant or animal cells.

On the other hand, spectinomycin, which is an antibotic derived from Streptomyces spectabilis, belongs to an aminoglycoside family and is composed of one sugar and two methyl groups originated from a methyl group donor, SAM.

DISCLOSURE OF THE INVENTION

Based on the fact that methyl groups of spectinomycin are derived from SAM, inventors of the present invention conducted intensive and thorough research into effects of SAM on biosynthesis of spectinomycin, resulting in the finding that SAM positively affects the biosynthesis of antibiotics, thereby increasing their production yield.

Therefore, it is an object of the present invention to provide an isolated nucleotide sequence encoding an enzyme catalyzing biosynthesis of SAM from Streptomyces spectabilis ATCC 27741 and an amino acid sequence translated from the isolated nucleotide sequence.

It is another object of the present invention to provide a method of increasing production of a useful secondary metabolite including antibiotics using SAM.

In accordance with the present invention, the first object is achieved by isolating a gene encoding an enzyme catalyzing SAM biosynthesis, which is derived from S. spectabilis, by obtaining a PCR product of 4.0 kb from a gene library of S. spectabilis using PCR, and confirming presence of a gene of about 1.2 kb in the PCR product encoding an enzyme catalyzing SAM biosynthesis, by sequencing the PCR product assaying activity of its translational product.

In accordance with the present invention, the second object is achieved by producing SAM, which is synthesized by the translational product of the isolated nucleotide sequence or that which is commercially available, having an ability to stimulate production of an antibiotic.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

FIG. 1 is a restriction map of a gene encoding an enzyme catalyzing SAM biosynthesis (SAM-s) carried in a recombinant plasmid, pJWK0012, which is originated from an E. coli plasmid;

FIG. 2 is a graph showing an ability of “SAM-s” to synthesize SAM, using BPLC, where commercially available SAM is used as a control;

FIG. 3 is a graph showing an effect of SAM produced by “SAM-s” on production yield of actinorhodin in S. lividans TK23 transformed with a gene encoding “SAM-s”;

FIG. 4 is a photograph showing an effect of SAM produced by “SAM-s” on production yield of actinorhodin in S. lividans TK23 treated with commercially available SAM; and

FIG. 5 is a graph showing an effect of SAM on production yield of undecylprodigiosin in S. lividans TK23.

BEST MODES FOR CARRYING OUT THE INVENTION

In accordance with the present invention, there is provided an enzyme catalyzing biosynthesis of S-adenosyl-L-methionine having an amino acid sequence consisting of the sequence shown in SEQ ID NO. 2, which is derived from Streptomyces spectabilis ATCC 27741.

In accordance with the present invention, there is provided an isolated nucleotide sequence consisting of the sequence shown in SEQ ID NO. 1, which encodes the amino acid sequence of SEQ ID NO. 2.

In accordance with the present invention, there is also provided a method of producing a useful secondary metabolite including antibiotics in a Streptomyces species-originated transformant into which a gene encoding an enzyme catalyzing biosynthesis of SAM is introduced, thereby improving productivity of the useful secondary metabolite.

In accordance with the present invention, there is further provided a method of producing a useful secondary metabolite including antibiotics by directly adding SAM to culture medium containing antibiotic-producing bacteria, thereby improving productivity of the useful secondary metabolite.

In the present invention, preferable examples of the useful secondary metabolite include antibiotics, such as polyketide antibiotics, anti-cancer agents, and vermicides.

In the present invention, a gene encoding an enzyme catalyzing SAM biosynthesis (SAM-s) is isolated from cosmid clones containing genes encoding enzymes stimulating biosynthesis of spectinomycin, which is obtained from a cDNA library of S. spectabilis ATCC 27741, where a 3.9 kb clone is primarily obtained. The nucleotide sequence of the 3.9 kb clone is partially determined by performing nucleotide sequencing, and its homology to known SAM synthetases, which mediate biosynthesis of SAM using ATP and methionine as substrates, is examined, based on the obtained nucleotide sequence, indicating the possible presence of a gene encoding an enzyme catalyzing SAM biosynthesis. The 3.9 kb clone is demonstrated to carry the gene encoding the enzyme catalyzing SAM biosynthesis by in vitro assaying the activity of its translated product.

In accordance with the present invention, a portion of the 3.9 kb clone, containing the gene encoding an enzyme catalyzing SAM biosynthesis (SAM-s), is introduced into Streptomyces species to assay production yield of an antibiotic, actinorhodin, and also, the effect of SAM on production of antibiotics is further investigated through direct treatment of cells with SAM, thereby demonstrating that SAM is effective in improving productivity of secondary metabolites including antibiotics.

In embodiments of the present invention, Streptomyces lividans TK23, which is commercially available, is transformed with the gene encoding “SAM-s”, and the resulting transformant exhibits mass production of actinorhodin, demonstrating that SAM is effective in enhancing antibiotic productivity of cells. Herein, application of SAM for high production of antibiotics is not limited to the transformant and the antibiotic, actinorhodin, but the effectiveness of SAM on production of antibiotics can be achieved with all Streptomyces species transformed with a gene encoding an enzyme catalyzing SAM biosynthesis.

In addition, when SAM is directly added to culture medium containing antibiotic-producing bacteria, productivity of antibiotics is increased 5 to 10 times, and especially, production of polyketide antibiotics is significantly increased.

The present invention will be explained in more detail with reference to the following examples in conjunction with the accompanying drawings. However, the following examples are provided only to illustrate the present invention, and the present invention is not limited to them.

EXAMPLE 1 Cloning of a Gene Encoding an Enzyme Capable of SAM Biosynthesis from Cosmid Clones of Streptomyces spectabilis ATCC 27741

Genes encoding enzymes catalyzing biosynthesis of an antibiotic are typically located together in a specific region of a genome. Therefore, there was used two cosmid clones harboring 30 to 40 kb fragment, which carries a gene family consisting of genes encoding enzymes participating in spectinomycin biosynthesis and may also include a gene encoding methyltransferase enzyme, one of enzymes mediating spectinomycin biosynthesis, which functions to transfer methyl groups. After digestion of the two cosmid clones with restriction enzymes, Southern Blotting was performed using metK gene, having high homology to methlytransferase at the nucleotide sequence level, as a probe.

As a result of Southern Blotting, a positive spot was observed, indicating a 3.9 kb fragment inserted into a BamHI site of pHCG121. 3.9 kb fragment was then subcloned into a BamHI site of pBluescript KS(+), giving a recombinant plasmid pHCG1647. From the subcloned 3.9 kb fragment, a 2.5 kb fragment, which is believed to carry a gene encoding an enzyme catalyzing SAM biosynthesis, was subcloned again into pBluescript KS(+) to form a recombinant plasmid pJWK0012.

EXAMPLE 2 Determination of Nucleotide Sequence of the Cloned 2.5 kb Fragment and its Corresponding Amino Acid Sequence

In order to determine a nucleotide sequence of the cloned 2.5 kb fragment and its corresponding amino acid sequence, the 2.5 kb insert carried in pJWK0012 prepared in the Example 1 was digested with restriction enzymes, ApaI, SalI and SacI, and then subcloned, followed by nucleotide sequencing. FIG. 1 shows a restriction map of the 2.5 kb fragment in pJWK0012 and its translational orientation.

Based on the nucleotide sequence of the 2.5 kb fragment, its amino acid sequence was obtained through search using a Codon Preference program (Bibb, M. J. et al., Gene, 1984). As a result, the 2.5 kb fragment was found to have an open reading frame consisting of a coding region ranging from nt 835 to nt 2051, which may express a protein consisting of 464 amino acid. The translational product of the open reading frame was, in the present invention, called “SAM-s”.

To investigate the homology of “SAM-s” to other known proteins, the amino acid sequence of “SAM-s” was compared to those of SAM synthetases of Streptomyces coelicolor, Bactillus subtilis and Escherichia coli, which were obtained from GeneBank DataBase (USA). It was found that “SAM-s” shares high homology with other synthetases. Also “SAM-s” of the present invention was found to have homology to some methyltransferases from microorganisms.

EXAMPLE 3 Assay for Activity of “SAM-s”

In order to analyze activity of “SAM-s”, the gene encoding “SAM-s” was expressed in E. coli, and the resulting translational product, “SAM-s”, was then isolated.

To express the gene encoding “SAM-s” in E. coli, the gene was inserted into a pET-21a vector, and then introduced into E. coli BL21. The expressed gene product, “SAM-s” was isolated using a His-Tag purification system. Thereafter, 10 to 50 μl of the enzyme solution containing the protein “SAM-s” was added to a reaction mixture containing 100 mM of Tris-HCl, 200 mM of KCl, 10 mM of MgCl₂, 1 mM DTT, 5 mM ATP, and 5 mM methionine, followed by incubation for 120 min at 30° C. After the incubation, reaction products were analyzed through HPLC using Reverse C18 column. In this regard, the column loaded with sample was initially equilibrated with a solution of 0.1 M of NaH₂PO₄/acetonitrile at a ratio of 98:2 (V/V), pH 2.65. Then, a second solution comprising 0.15 M NaH₂PO₄/acetonitrile at a ratio of 74:26 (V/V) was applied with continuous mixing with the first solution, forming a concentration gradient.

As shown in FIG. 2, the product of the catalytic activity of the protein expressed in E. coli is proven to be SAM. That is, when the expressed protein is supplied with ATP and methionine as substrates, the product has an HPLC retention time identical to commercially available SAM, indicating that the protein expressed in E. coli has an activity to synthesize SAM using ATP and methionine as substrates.

The gene encoding “SAM-s” was first inserted into pWHM3, which is a shuttle vector between E. coli and Streptomyces species, giving an expression vector pSAM-s. The plasmid pSAM-s was then introduced into S. lividans TK23. The resulting transformant, Streptomyces lividans TK-23 harboring pSAM-s, was deposited in the Korean Culture Center of Microorganisms with accession No. KCCM 10397 on Jul. 2, 2002. The transformant was incubated in one liter of a medium including 50 g of glycerol, 5 g of glutamic acid, 21 g of morpholinopropane sulfonic acid, 200 mg of MgSO₄7H₂O, 100 mg of CaCl₂2H₂O, 100 mg NaCl, 82 mg KH₂PO₄, 9 mg FeSO₄7H₂O, and 2 ml of trace element solution, adjusted to pH 6.5. During incubation for 7 days at 28° C., production yield of actinorhodin, which is a main antibiotic produced from S. lividans TK23, was analyzed. The results are shown in FIG. 4.

As apparent in FIG. 3, when SAM was over-produced in S. lividans TK 23 through over-expression of “SAM-s”, it was observed that production of actinorhodin in the transformant was enhanced to over six times in comparison with that of a wild type S. lividans.

EXAMPLE 5 Effect of Externally Added SAM on Productivity of Actinorhodin

Based on the finding that in vivo over-expressed SAM positively affects production yield of actinorhodin in S. lividans TK23, an effect of SAM on productivity of actinorhodin was investigated when commercially available SAM is added directly to culture medium containing S. lividans TK23. As such, wild type S. lividans TK23 was treated with 1 mM of commercially available SAM.

The result is shown in FIG. 4, where actinorhodin produced in S. lividans TK23 treated with SAM, and the control not treated with SAM, indicated by a blue color. As shown in FIG. 4, it was found that S. lividans TK23 treated with SAM produces more actinorhodin than S. lividans TK23 not treated with SAM, demonstrating that SAM positively affects productivity of actinorhodin.

EXAMPLE 6 Effect of SAM on Productivity of Undecylprodigiosin in S. lividans TK23

S. lividans TK23 transformed with the vector pSAM-s was incubated under the same culture conditions as those used for production of actinorhodin. To determine the amount of undecylprodigiosin produced, after adjusting pH to 12, absorbance was measured at 468 nm, and concentration of the antibiotic was calculated according to the following formula: concentration=OD value×9.4673.

As apparent FIG. 5, productivity of undecylprodigiosin was very high in comparison with a control not transformed with the vector pSAM-s, indicating that SAM positively affects production yield of undecylprodigiosin.

EXAMPLES 7 to 13 Effect of SAM on Productivity of Antibiotics in Streptomyces species

TABLE 1 Culture medium and culture condition Streptomyces Culture medium (/L) and Exp. Antibiotic sp. culture condition 7 Avermectin S. avermitilis 15 g glucose, 0.5 g asparagine, 0.5 g K₂HPO₄, pH 7.0, 25° C., incubation for 5 days 8 Monensin S. cinnamonensis 2.5% glucose, 1.5% soybean meal, 0.3% CaCO₃, 0.03% FeSO₄7H₂O, 0.003% MnCl₂4H₂O, pH 7.0, 30° C., incubation for 5 days 9 Spectinomycin S. spectabilis 10 g Maltose, 5 g tryptone, 1 g K₂HPO₄, 2 g NaCl, pH7.0, 30° C., incubation for 5 days 10 Doxorubicin S. peucetius 60 g Glucose, 8 g yeast extract, 20 g malt extract, 2 g NaCl, 15 g MOPS sodium salt, 0.1 g MgSO₄, 0.01 g FeSO₄7H₂O, 0.01 g ZnSO₄7H₂O, pH 7.0, 30° C., incubation for 5 days 11 Streptomycin S. griseus 1% glucose, 0.4% peptone, 0.2% meat extract, 0.2% yeast extract, 0.5% NaCl, 0.025% MgSO₄7H₂O, pH 7, 30° C., incubation for 5 days 12 Tetracyclin S. aureofaciens 3% corn flour, 4% corn steep liquor, 5% corn starch, 0.7% (NH₄)₂SO₄, 0.1% NH₄Cl, 5 ppm CoCl₂, 0.9% CoSO₃, 2% rice bran oil, pH7, 28° C., incubation for 5 days 13 Chlortetracyclin S. aureofaciens 1% sucrose, 1% corn steep liquor, 0.2% (NH₄)₂HPO₄, 0.2% KH₂PO₄, 0.1% CaCO₃, 0.025% MgSO₄7H₂O, 0.005% ZnSO₄7H₂O, 0.00033% CuSO₄5H₂O, 0.00033% MnCl₂4H₂O, incubation for 5 days

Each Streptomyces species was incubated in its corresponding culture medium according to Table 1, and treated with 1 mM of SAM (Sigma, USA). After incubation for 5 days, antibiotic concentration was measured in each culture medium. The results are given in Table 2, below. It was found that each Streptomyces species, as a treatment group, produces a much higher amount of its specific antibiotic than a control group not treated with SAM.

TABLE 2 Production amount of an antibiotic in Streptomyces sp. treated or not treated with SAM Production Production amount of a amount of a control treatment Streptomyces group group Exp. Antibiotic sp. (μg/ml) (μg/ml) 7 Avermectin S. avermitilis 5 25 8 Monensin S. cinnamonensis 30 180 9 Spectinomycin S. spectabilis 5 35 10 Doxorubicin S. peucetius 38 300 11 Streptomycin S. griseus 101 602 12 Tetracycline S. aureofaciens 30 188 13 Chlortetracycline S. aureofaciens 25 130

As shown in Table 2, Streptomyces species treated with SAM produced 5 to 10 times more antibiotic than the control, indicating that SAM positively affects production yield of various antibiotics.

INDUSTRIAL APPLICABILITY

As described hereinbefore, the present invention provides an isolated nucleotide sequence of a gene encoding an enzyme catalyzing biosynthesis of SAM, which is derived from Streptomyces spectabilis ATCC 27741, and its amino acid sequence. SAM, which is produced by the enzyme of the present invention or purchased commercially, is very effective in increasing productivity of various antibiotics. Therefore, the isolated nucleotide sequence of the present invention is capable of being utilized in mass production of secondary metabolites including antibiotics, and thus is very useful in pharmaceutical industries. 

1. A method for production of an antibiotic comprising the steps: i) producing S-adenosyl-L-methionine by transforming a Streptomyces species selected from the group consisting of Streptomyces spectabilis, Streptomyces lividans, Streptomyces avermitilis, Streptomyces cinnamonenesis, Streptomyces peucetius, Streptomyces griseus, and Streptomyces aureofaciens with an expression vector comprising a polynucleotide obtained from Streptomyces spectabilis ATCC 27741 encoding a polypeptide having the enzymatic activity of catalyzing the biosynthesis of said S-adenosyl-L-methionine, wherein said polypeptide has the amino acid sequence of SEQ ID NO: 2; and ii) culturing said transformed Streptomyces species producing said S-adenosyl-L-methionine in an antibiotic culture medium for the mass production of antibiotics, wherein said antibiotics are avermectin, monensin, spectinomycin, doxorubicin, streptomycin, tetracycline, chlortetracycline, actinorhodin or undecylprodigiosin.
 2. The method for mass production of claim 1, wherein the Streptomyces lividians is Streptomyces lividians TK23.
 3. The method for mass production of claim 2, wherein the Streptomyces lividians TK23 has an accession No. KCCM 10397 (Korean Culture Center of Microorganisms). 